Multimodal neuroimaging with optically pumped magnetometers: A simultaneous MEG-EEG-fNIRS acquisition system.

Multimodal neuroimaging plays an important role in neuroscience research. Integrated noninvasive neuroimaging modalities, such as magnetoencephalography (MEG), electroencephalography (EEG) and functional near-infrared spectroscopy (fNIRS), allow neural activity and related physiological processes in the brain to be precisely and comprehensively depicted, providing an effective and advanced platform to study brain function. Noncryogenic optically pumped magnetometer (OPM) MEG has high signal power due to its on-scalp sensor layout and enables more flexible configurations than traditional commercial superconducting MEG. Here, we integrate OPM-MEG with EEG and fNIRS to develop a multimodal neuroimaging system that can simultaneously measure brain electrophysiology and hemodynamics. We conducted a series of experiments to demonstrate the feasibility and robustness of our MEG-EEG-fNIRS acquisition system. The complementary neural and physiological signals simultaneously collected by our multimodal imaging system provide opportunities for a wide range of potential applications in neurovascular coupling, wearable neuroimaging, hyperscanning and brain-computer interfaces.

Automatic coregistration of MRI and on-scalp MEG.

BACKGROUND: Recent progress in optically pumped magnetometers (OPMs) and high-temperature superconducting quantum interference devices (SQUIDs) has facilitated the development of an on-scalp magnetoencephalography (MEG) system that offers high signal intensity and flexibility at a lower cost. While the on-scalp sensor array has high flexibility, it brings new challenges to accurate sensor-to-brain coregistration, which is essential for MEG source localization. NEW METHOD: A novel automatic filtering algorithm based on plane segmentation was proposed to locate on-scalp MEG sensors in 3D images reconstructed from optical scanning. Global image registration was employed for the automatic alignment of anatomical images and sensor positions. RESULTS: Seventy-one sensor dummies on the scalp were located and registered to brain anatomical images. The deviations of the sensor location and orientation from the averaged result of 10 measurements were less than 1 mm and 0.6°, respectively. The entire process could be completed in less than 4 min. COMPARISON WITH EXISTING METHODS: Compared with existing methods that involve various manual procedures, such as moving digitizers to fiducials and repeatedly pulling out sensors, our proposed coregistration method is more efficient and accurate. CONCLUSION: An automatic method for the coregistration of anatomical structure and on-scalp sensors that will have a large impact on the practical use of on-scalp MEG is developed.

Neural representations of imagined speech revealed by frequency-tagged magnetoencephalography responses.

Speech mental imagery is a quasi-perceptual experience that occurs in the absence of real speech stimulation. How imagined speech with higher-order structures such as words, phrases and sentences is rapidly organized and internally constructed remains elusive. To address this issue, subjects were tasked with imagining and perceiving poems along with a sequence of reference sounds with a presentation rate of 4 Hz while magnetoencephalography (MEG) recording was conducted. Giving that a sentence in a traditional Chinese poem is five syllables, a sentential rhythm was generated at a distinctive frequency of 0.8 Hz. Using the frequency tagging we concurrently tracked the neural processing timescale to the top-down generation of rhythmic constructs embedded in speech mental imagery and the bottom-up sensory-driven activity that were precisely tagged at the sentence-level rate of 0.8 Hz and a stimulus-level rate of 4 Hz, respectively. We found similar neural responses induced by the internal construction of sentences from syllables with both imagined and perceived poems and further revealed shared and distinct cohorts of cortical areas corresponding to the sentence-level rhythm in imagery and perception. This study supports the view of a common mechanism between imagery and perception by illustrating the neural representations of higher-order rhythmic structures embedded in imagined and perceived speech.

Enhanced Fast-VESTAL for Magnetoencephalography Source Imaging: From Theory to Clinical Application in Epilepsy.

A novel magnetoencephalography source imaging approach called Fast Vector-based Spatio-Temporal Analysis (Fast-VESTAL) has been successfully applied in creating source images from evoked and resting-state data from both healthy subjects and individuals with neurological and/or psychiatric disorders, but its reconstructed source images may show false-positive activations, especially under low signal-to-noise ratio conditions. Here, to effectively reduce false-positive artifacts, we introduced an enhanced Fast-VESTAL (eFast-VESTAL) approach that adopts generalized second-order cone programming. We compared the spatiotemporal characteristics of the eFast-VESTAL approach to those of the popular distributed source approaches (e.g., the minimum L2-norm/ mixed-norm methods) using computer simulations and auditory experiments. More importantly, we applied eFast-VESTAL to the presurgical evaluation of epilepsy. Our results demonstrated that eFast-VESTAL exhibited a lower dipole localization error and/or a higher correlation coefficient (CC) between the estimated source time series and ground truth under various conditions of source waveforms. Experimentally, eFast-VESTAL displayed more focal activation maps and a higher CC between the raw and predicted sensor data in response to auditory stimulation. Notably, eFast-VESTAL was the most accurate method for noninvasively detecting the epileptic zones determined using more invasive stereo-electroencephalography in the comparison.

EMS-Net: A Deep Learning Method for Autodetecting Epileptic Magnetoencephalography Spikes.

Epilepsy is a neurological disorder characterized by sudden and unpredictable epileptic seizures, which incurs significant negative impacts on patients' physical, psychological and social health. A practical approach to assist with the clinical assessment and treatment planning for patients is to process magnetoencephalography (MEG) data to identify epileptogenic zones. As a widely accepted biomarker of epileptic foci, epileptic MEG spikes need to be precisely detected. Given that the visual inspection of spikes is time consuming, an automatic and efficient system with adequate accuracy for spike detection is valuable in clinical practice. However, current approaches for MEG spike autodetection are dependent on hand-engineered features. Here, we propose a novel multiview Epileptic MEG Spikes detection algorithm based on a deep learning Network (EMS-Net) to accurately and efficiently recognize the spike events from MEG raw data. The results of the leave-k-subject-out validation tests for multiple datasets (i.e., balanced and realistic datasets) showed that EMS-Net achieved state-of-the-art classification performance (i.e., accuracy: 91.82% - 99.89%; precision: 91.90% - 99.45%; sensitivity: 91.61% - 99.53%; specificity: 91.60% - 99.96%; f1 score: 91.70% - 99.48%; and area under the curve: 0.9688 - 0.9998).

Neural tracking of speech mental imagery during rhythmic inner counting.

The subjective inner experience of mental imagery is among the most ubiquitous human experiences in daily life. Elucidating the neural implementation underpinning the dynamic construction of mental imagery is critical to understanding high-order cognitive function in the human brain. Here, we applied a frequency-tagging method to isolate the top-down process of speech mental imagery from bottom-up sensory-driven activities and concurrently tracked the neural processing time scales corresponding to the two processes in human subjects. Notably, by estimating the source of the magnetoencephalography (MEG) signals, we identified isolated brain networks activated at the imagery-rate frequency. In contrast, more extensive brain regions in the auditory temporal cortex were activated at the stimulus-rate frequency. Furthermore, intracranial stereotactic electroencephalogram (sEEG) evidence confirmed the participation of the inferior frontal gyrus in generating speech mental imagery. Our results indicate that a disassociated neural network underlies the dynamic construction of speech mental imagery independent of auditory perception.

A high-performance compact magnetic shield for optically pumped magnetometer-based magnetoencephalography.

The rapid development of the optically pumped magnetometer (OPM) has offered a much more flexible method for magnetoencephalography (MEG). Without using liquid helium and its associated dewar device in the OPM detectors, the large and expensive magnetically shielded room (MSR) for traditional MEG systems could be replaced by a compact shield. In the present work, an economic and compact cylindrical shield was designed and built to meet the low-field working requirement of the OPM in detecting human brain neuronal activities. The performance of the compact shield was evaluated and further compared with that of a commercial MSR. Our results showed that the residual magnetic fields and background noise of the compact shield were lower than or comparable to those of the MSR. The remnant field in the shield is found to be 4.2 nT, a factor of 13 000 smaller than the geomagnetic field which is applied to the transverse direction of the shield, and the longitudinal shielding factors measured using a known alternating-current magnetic field are approximately 191, 205, and 3130 at 0.1 Hz, 1 Hz, and 10 Hz, respectively; in addition, the evoked dynamic waveforms in the human auditory cortex that were recorded separately in these two shields demonstrated consistency. Our findings suggested that a compact shield is feasible for OPM-based MEG applications with high performance and low cost.

The Cortical Maps of Hierarchical Linguistic Structures during Speech Perception.

The hierarchical nature of language requires human brain to internally parse connected-speech and incrementally construct abstract linguistic structures. Recent research revealed multiple neural processing timescales underlying grammar-based configuration of linguistic hierarchies. However, little is known about where in the whole cerebral cortex such temporally scaled neural processes occur. This study used novel magnetoencephalography source imaging techniques combined with a unique language stimulation paradigm to segregate cortical maps synchronized to 3 levels of linguistic units (i.e., words, phrases, and sentences). Notably, distinct ensembles of cortical loci were identified to feature structures at different levels. The superior temporal gyrus was found to be involved in processing all 3 linguistic levels while distinct ensembles of other brain regions were recruited to encode each linguistic level. Neural activities in the right motor cortex only followed the rhythm of monosyllabic words which have clear acoustic boundaries, whereas the left anterior temporal lobe and the left inferior frontal gyrus were selectively recruited in processing phrases or sentences. Our results ground a multi-timescale hierarchical neural processing of speech in neuroanatomical reality with specific sets of cortices responsible for different levels of linguistic units.

Frequency-dependent tACS modulation of BOLD signal during rhythmic visual stimulation.

Transcranial alternating current stimulation (tACS) has emerged as a promising tool for modulating cortical oscillations. In previous electroencephalogram (EEG) studies, tACS has been found to modulate brain oscillatory activity in a frequency-specific manner. However, the spatial distribution and hemodynamic response for this modulation remains poorly understood. Functional magnetic resonance imaging (fMRI) has the advantage of measuring neuronal activity in regions not only below the tACS electrodes but also across the whole brain with high spatial resolution. Here, we measured fMRI signal while applying tACS to modulate rhythmic visual activity. During fMRI acquisition, tACS at different frequencies (4, 8, 16, and 32 Hz) was applied along with visual flicker stimulation at 8 and 16 Hz. We analyzed the blood-oxygen-level-dependent (BOLD) signal difference between tACS-ON vs tACS-OFF, and different frequency combinations (e.g., 4 Hz tACS, 8 Hz flicker vs 8 Hz tACS, 8 Hz flicker). We observed significant tACS modulation effects on BOLD responses when the tACS frequency matched the visual flicker frequency or the second harmonic frequency. The main effects were predominantly seen in regions that were activated by the visual task and targeted by the tACS current distribution. These findings bridge different scientific domains of tACS research and demonstrate that fMRI could localize the tACS effect on stimulus-induced brain rhythms, which could lead to a new approach for understanding the high-level cognitive process shaped by the ongoing oscillatory signal.

EEG/MEG source imaging using fMRI informed time-variant constraints.

Multimodal functional neuroimaging by combining functional magnetic resonance imaging (fMRI) and electroencephalography (EEG) or magnetoencephalography (MEG) is able to provide high spatiotemporal resolution mapping of brain activity. However, the accuracy of fMRI-constrained EEG/MEG source imaging may be degraded by potential spatial mismatches between the locations of fMRI activation and electrical source activities. To address this problem, we propose a novel fMRI informed time-variant constraint (FITC) method. The weights in FITC are determined by combining the fMRI activities and electrical source activities in a time-variant manner to reduce the impact of the fMRI extra sources. The fMRI weights are modified using cross-talk matrix and normalized partial area under the curve to reduce the impact of fMRI missing sources. Monte Carlo simulations were performed to compare the source estimates produced by L2-minimum norm estimation (MNE), fMRI-weighted minimum norm estimation (fMNE), FITC, and depth-weighted FITC (wFITC) algorithms with various spatial mismatch conditions. Localization error and temporal correlation were calculated to compare the four algorithms under different conditions. The simulation results indicated that the FITC and wFITC methods were more robust than the MNE and fMNE algorithms. Moreover, FITC and wFITC were significantly better than fMNE under the fMRI missing sources condition. A human visual-stimulus EEG, MEG, and fMRI test was performed, and the experimental data revealed that FITC and wFITC displayed more focal areas than fMNE and MNE. In conclusion, the proposed FITC method is able to better resolve the spatial mismatch problems encountered in fMRI-constrained EEG/MEG source imaging.

Magnetoencephalography with a Cs-based high-sensitivity compact atomic magnetometer.

In recent years, substantial progress has been made in developing a new generation of magnetoencephalography (MEG) with a spin-exchange relaxation free (SERF)-based atomic magnetometer (AM). An AM employs alkali atoms to detect weak magnetic fields. A compact AM array with high sensitivity is crucial to the design; however, most proposed compact AMs are potassium (K)- or rubidium (Rb)-based with single beam configurations. In the present study, a pump-probe two beam configuration with a Cesium (Cs)-based AM (Cs-AM) is introduced to detect human neuronal magnetic fields. The length of the vapor cell is 4 mm, which can fully satisfy the need of designing a compact sensor array. Compared with state-of-the-art compact AMs, our new Cs-AM has two advantages. First, it can be operated in a SERF regime, requiring much lower heating temperature, which benefits the sensor with a closer distance to scalp due to ease of thermal insulation and less electric heating noise interference. Second, the two-beam configuration in the design can achieve higher sensitivity. It is free of magnetic modulation, which is necessary in one-beam AMs; however, such modulation may cause other interference in multi-channel circumstances. In the frequency band between 10 Hz and 30 Hz, the noise level of the proposed Cs-AM is approximately 10 f T/Hz1/2, which is comparable with state-of-the-art K- or Rb-based compact AMs. The performance of the Cs-AM was verified by measuring human auditory evoked fields (AEFs) in reference to commercial superconducting quantum interference device (SQUID) channels. By using a Cs-AM, we observed a clear peak in AEFs around 100 ms (M100) with a much larger amplitude compared with that of a SQUID, and the temporal profiles of the two devices were in good agreement. The results indicate the possibility of using the compact Cs-AM for MEG recordings, and the current Cs-AM has the potential to be designed for multi-sensor arrays and gradiometers for future neuroscience studies.

MR imaging of oscillatory magnetic field changes: Progressing from phantom to human.

Detection of ultra-weak oscillatory magnetic field changes using MRI is of great research interest not only for neuronal current MRI of endogenous neuronal oscillations but also for direct visualization of exogenous transcranial currents or iron oxide contrast agent distribution. In this work, we present a novel oscillatory-selective detection (OSD) method that is magnitude-sensitive to the oscillatory magnetic field changes and immune to the main field inhomogeneity. In OSD, a train of 180° pulses with alternating polarity and mirror symmetry are used to refocus and accumulate magnetization changes induced by external oscillatory fields. After taking both the signal change and image signal-to-noise ratio (SNR) into account, a final 90° pulse with a phase offset of 45° is applied to store a combination of the current-induced signal change and background magnetization for the subsequent EPI acquisition. Its performance was demonstrated in phantom and human studies, both of which showed much better detection in the comparison with the recently proposed spin-lock oscillatory excitation (SLOE) method. OSD was further successfully applied in imaging transcranial alternating current stimulation (tACS) induced field changes in the human brain. These promising results suggest that OSD can overcome the limitation of field inhomogeneity impeding previous oscillatory current MRI sensitivity and be a viable tool in future tACS study.

A comprehensive study of sensitivity in measuring oscillatory magnetic fields using rotary saturation pulse sequences.

Detecting the oscillatory currents with a specific frequency distribution may have the potential to make neuronal current MRI (ncMRI) come true. The phase shift or dephasing induced by both positive and negative episodes of oscillatory neuronal currents is likely to be canceled out over the echo time in typical BOLD-contrast fMRI experiments. Based on the contrast of rotary saturation, both of the recently developed spin-locked oscillatory excitation (SLOE) and stimulus-induced rotary saturation (SIRS) pulse sequences have been demonstrated to be able to detect weak oscillatory magnetic fields in phantoms with 3T MR scanners. In this report, through Bloch equation simulation as well as water phantom and anesthetic rats experiments, we comprehensively evaluate and compare the sensitivities of these two methods (SLOE and SIRS) in detecting the oscillatory magnetic fields for both high (100 Hz) and low (10 Hz) oscillation frequencies, while using their respective optimal imaging parameters. In agreement with the theoretical predications, both the simulated and experimental results showed that the SLOE method features a much higher detection sensitivity of weak magnetic fields than that of the SIRS method. SLOE was able to detect applied oscillatory magnetic fields as low as 0.1 nT in a water phantom and 0.5 nT in rat brains and the deteriorated noise levels in rat data may account for the reduced sensitivity in vivo. These promising results form the foundation for direct detection of in vivo neuronal currents using MRI.

Detection of subnanotesla oscillatory magnetic fields using MRI.

PURPOSE: Direct mapping of neuronal currents using MRI would have fundamental impacts on brain functional imaging. Previous reports indicated that the stimulus-induced rotary saturation (SIRS) mechanism had the best potential of direct detection of neural oscillations; however, it lacked the high-sensitivity level needed. In this study, a novel strategy is proposed in an effort to improve the detection sensitivity. METHODS: In our modified SIRS sequence, an external oscillatory magnetic field is used as the excitation pulse in place of the standard 90-degree excitation pulse. This approach could potentially lead to tens or even hundreds times of enhancement in the detection sensitivity for low field signals. It also helps to lower the physiological noise, allows for shorter pulse repetition time, and is less affected by the blood oxygen level. RESULTS: We demonstrate that a 100-Hz oscillatory magnetic field with magnitude as low as 0.25 nanotesla generated in a current loop can be robustly detected using a 3-Tesla MRI scanner. CONCLUSION: The modified SIRS sequence offers higher detection sensitivity as well as several additional advantages. These promising results suggest that the direct detection of neural oscillation might be within the grasp of the current MRI technology.

Re-trafficking of hERG reverses long QT syndrome 2 phenotype in human iPS-derived cardiomyocytes.

AIMS: Long QT syndrome 2 (LQTS2) caused by missense mutations in hERG channel is clinically associated with abnormally prolonged ventricular repolarization and sudden cardiac deaths. Modelling monogenic arrhythmogenic diseases using human-induced pluripotent stem cells (hiPSCs) offers unprecedented mechanistic insights into disease pathogenesis. We utilized LQTS2-hiPSC-derived cardiomyocytes (CMs) to elucidate pathological changes and to demonstrate reversal of LQTS2 phenotype in a therapeutic intervention using a pharmacological agent, (N-[N-(N-acetyl-l-leucyl)-l-leucyl]-l-norleucine) (ALLN). METHODS AND RESULTS: We generated LQTS2-specific CMs (A561V missense mutation in KCNH2) from iPSCs using the virus-free reprogramming method. These CMs recapitulate dysfunction of hERG potassium channel with diminished IKr currents, prolonged repolarization durations, and elevated arrhythmogenesis due to reduced membrane localization of glycosylated/mature hERG. Dysregulated expression of folding chaperones and processing proteasomes coupled with sequestered hERG in the endoplasmic reticulum confirmed trafficking-induced disease manifestation. Treatment with ALLN, not only increased membrane localization of mature hERG but also reduced repolarization, increased IKr currents and reduced arrhythmogenic events. Diverged from biophysical interference of hERG channel, our results show that modulation of chaperone proteins could be therapeutic in LQTS2 treatment. CONCLUSION: Our in vitro study shows an alternative approach to rescue diseased LQTS2 phenotype via corrective re-trafficking therapy using a small chemical molecule, such as ALLN. This potentially novel approach may have ramifications in other clinically relevant trafficking disorders.

Electrophysiology of human cardiac atrial and ventricular telocytes.

Telocytes (TCs) with exceptionally long cellular processes of telopodes have been described in human epicardium to act as structural supporting cells in the heart. We examined myocardial chamber-specific TCs identified in atrial and ventricular fibroblast culture using immunocytochemistry and studied their electrophysiological property by whole-cell patch clamp. Atrial and ventricular TCs with extended telopodes and alternating podoms and podomers that expressed CD34, c-Kit and PDGFR-ß were identified. These cells expressed large conductance Ca²âº-activated K⁺ current (BK(Ca)) and inwardly rectifying K⁺ current (IK(ir)), but not transient outward K⁺ current (I(to)) and ATP-sensitive potassium current (K(ATP)). The active channels were functionally competent with demonstrated modulatory response to H2 S and transforming growth factor (TGF)-ß1 whereby H2S significantly inhibited the stimulatory effect of TGF-ß1 on current density of both BKCa and IK(ir). Furthermore, H2S attenuated TGF-ß1-stimulated KCa1.1/Kv1.1 (encode BK(Ca)) and Kir2.1 (encode IK(ir)) expression in TCs. Our results show that functionally competent K⁺ channels are present in human atrial and ventricular TCs and their modulation may have significant implications in myocardial physiopathology.

Hydrogen sulphide suppresses human atrial fibroblast proliferation and transformation to myofibroblasts.

Cardiac fibroblasts are crucial in pathophysiology of the myocardium whereby their aberrant proliferation has significant impact on cardiac function. Hydrogen sulphide (H2S) is a gaseous modulator of potassium channels on cardiomyocytes and has been reported to attenuate cardiac fibrosis. Yet, the mechanism of H2S in modulating proliferation of cardiac fibroblasts remains poorly understood. We hypothesized that H2S inhibits proliferative response of atrial fibroblasts through modulation of potassium channels. Biophysical property of potassium channels in human atrial fibroblasts was examined by whole-cell patch clamp technique and their cellular proliferation in response to H2S was assessed by BrdU assay. Large conductance Ca(2+)-activated K(+) current (BK(Ca)), transient outward K(+) current (I(to)) and inwardly rectifying K(+) current (IK(ir)) were found in human atrial fibroblasts. Current density of BK(Ca) (IC50 = 69.4 µM; n = 6), I(to) (IC50 = 55.1 µM; n = 6) and IK(ir) (IC50 = 78.9 µM; n = 6) was significantly decreased (P < 0.05) by acute exposure to NaHS (a H2S donor) in atrial fibroblasts. Furthermore, NaHS (100-500 µM) inhibited fibroblast proliferation induced by transforming growth factor-ß1 (TGF-ß1; 1 ng/ml), Ang II (100 nM) or 20% FBS. Pre-conditioning of fibroblasts with NaHS decreased basal expression of Kv4.3 (encode I(to)), but not KCa1.1 (encode BK(Ca)) and Kir2.1 (encode IK(ir)). Furthermore, H2S significantly attenuated TGF-ß1-stimulated Kv4.3 and α-smooth muscle actin expression, which coincided with its inhibition of TGF-ß-induced myofibroblast transformation. Our results show that H2S attenuates atrial fibroblast proliferation via suppression of K(+) channel activity and moderates their differentiation towards myofibroblasts.

Hydrogen sulfide suppresses outward rectifier potassium currents in human pluripotent stem cell-derived cardiomyocytes.

AIM: Hydrogen sulfide (H2S) is a promising cardioprotective agent and a potential modulator of cardiac ion currents. Yet its cardiac effects on humans are poorly understood due to lack of functional cardiomyocytes. This study investigates electrophysiological responses of human pluripotent stem cells (hPSCs) derived cardiomyocytes towards H2S. METHODS AND RESULTS: Cardiomyocytes of ventricular, atrial and nodal subtypes differentiated from H9 embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) were electrophysiologically characterized. The effect of NaHS, a donor of H2S, on action potential (AP), outward rectifier potassium currents (I(Ks) and I(Kr)), L-type Ca²âº currents (I(CaL)) and hyperpolarization-activated inward current (I(f)) were determined by patch-clamp electrophysiology and confocal calcium imaging. In a concentration-dependent manner, NaHS (100 to 300 µM) consistently altered the action potential properties including prolonging action potential duration (APD) and slowing down contracting rates of ventricular-and atrial-like cardiomyocytes derived from both hESCs and hiPSCs. Moreover, inhibitions of slow and rapid I(K) (I(Ks) and I(Kr)), I(CaL) and I(f) were found in NaHS treated cardiomyocytes and it could collectively contribute to the remodeling of AP properties. CONCLUSIONS: This is the first demonstration of effects of H2S on cardiac electrophysiology of human ventricular-like, atrial-like and nodal-like cardiomyocytes. It reaffirmed the inhibitory effect of H2S on I(CaL) and revealed additional novel inhibitory effects on I(f), I(Ks) and I(Kr) currents in human cardiomyocytes.

Angiotensin II decreases spontaneous firing rate of guinea-pig sino-atrial node cells.

Angiotensin II (Ang II) plays an important role in the regulation of cardiac function, but its electrophysiological effects on sino-atrial (SA) node are not well understood. In this study, the immediate effect of Ang II on action potentials and ionic currents were investigated by using whole-cell patch-clamps in single guinea-pig SA node pacemaker cells. We demonstrated that Ang II exerted a negative effect on spontaneous firing rate, with a concomitant reduction in the slope of diastolic depolarization. The inhibitory effect of Ang II on spontaneous activity displayed a concentration-dependent manner in the range of 1-1000 nM, with IC50 of 8.34 nM. Ang II type 1 (AT1) receptor antagonist valsartan (1 µM) abolished the inhibitory effect. In contrast, Ang II type 2 (AT2) receptor antagonist, PD123319 (1 µM) didn't affect the action of Ang II. Ang II had no significant effect on hyperpolarization-activated current (If) in SA node cells. However, it significantly slowed the deactivation of the slowly activated delayed rectifier K+ current (Iks) and increased the tail current density. Furthermore, Ang II decreased the current density of L-type Ca2+ current in SA node cells. Our data demonstrate that Ang II reduces the auto rhythm of SA node cells via enhancing Iks and reducing ICaL. The result suggests a potential mechanism by which elevated levels of Ang II may be involved in the occurrence of SA node dysfunction in cardiac pathophysiology.

Identification and characterization of a novel cell-penetrating peptide.

Cell-penetrating peptides (CPPs) are short amino acid sequences that promote their own translocation across cell plasma membrane. When linked with cargo such as polypeptides, nucleic acid, or liposomes, CPPs can facilitate the transport of these entities across the cell membrane. Therefore, CPPs are receiving increased interest in drug delivery and gene therapy. The majority of CPPs identified so far are polycationic peptides which interact with heparin sulfate chains of plasma membrane for internalization. Here, we report the identification and characterization of a conformationally constrained 13 amino acid peptide (CVQWSLLRGYQPC, designated as S41) which is clearly distinct from classical polycationic peptides. Immunofluorescence assay was employed to test the cellular uptake of S41 in mouse neuroblastoma cell line Neuro2A (N2A) and rat cerebellar granule neurons (CGNs). Internalization of S41 was further examined in N2A cells by means of mutational analysis, flow cytometry and confocal microscopy. Our results demonstrate that S41 can enter cells through lipid rafts dependent endocytosis.